The invention relates to peptides, to antibodies produced with their help and to the use of such peptides and antibodies for therapeutic and diagnostic purposes.
The organism is protected from the loss of blood by the coagulation system. The coagulation cascade leads to the activation of the protease thrombin, which converts fibrinogen to fibrin by eliminating the A and B fibrino-peptides. The individual fibrin molecules aggregate to each other (so-called xe2x80x9csoft clotxe2x80x9d) and are then normally crosslinked to each other by the transpeptidase factor XIII (so-called xe2x80x9chard clotxe2x80x9d). This wound closure is lysed by the fibrinolytic system, which is activated in a counteractive manner. The key enzyme of fibrinolysis is the protease plasmin, which essentially cleaves fibrinogen and fibrin into the D and E fragments. Fibrinogen is constructed symmetrically from 2 tripeptides, which are linked to each other by means of disulfide bridges in the vicinity of the N termini. When fibrin or fibrinogen is cleaved, 1 molecule of fragment E, which comprises the central linkage region of the fibrin(ogen) molecule, and 2 molecules of fragment D are therefore produced per molecule. In a hard clot, the D domains of the fibrin are crosslinked, so that degradation by plasmin liberates D dimer and fragment E. The E fragment itself is subjected to two further degradation steps. In its first and second form (E1 and E2, respectively), it is bound non-covalently to D dimer and forms the DD/E complex. It is only after the second enzymatic degradation step that the E3 fragment dissociates from the D dimer molecule.
Proteins which carry several immunochemically identical epitopes within one molecule or proteins, such as the D dimer resulting from the cleavage of fibrin or fibrinogen gen, which, at least under physiological conditions, are constituted as oligomers of protein molecules which in each case carry at least one immunochemically identical epitope, are also designated xe2x80x9cintramolecular oligomersxe2x80x9d.
An unwanted activation of the coagulation system can take place in the vascular system in many pathological situations, resulting in subsequent occlusion. This can lead to serious heart attacks and thrombo-embolisms. For the purposes of supervising the therapy in patients who are being treated with thrombolytic agents on account of these hypercoagulatory conditions, the success of the lysis must be monitored. This is done by determining the D dimer. However, the thrombolytic agents are not specific, so that fibrinogen can also be degraded to an increased extent as a result of systemic activation of the plasmin. It would be possible to detect this degradation of fibrinogen in a timely manner by determining the E fragment. Fibrinogen is also predominantly degraded in hyperfibrinolytic conditions, triggered, for instance, in sepsis by way of the complement system, which can lead, for example, to the development of disseminated intravascular coagulation (DIC). However, consumption of fibrinogen carries with it an increased risk of bleeding, which risk can thus be recognized diagnostically in a timely manner by determining fragment E, and thereby counter- acted therapeutically.
Numerous methods are known for detectingdegradation products of fibrin(ogen), such as, for example, the hemaglutination inhibition test (Mersky C. et al., xe2x80x9cA rapid, simple, sensitive method for measuring fibrinolytic split products in human serumxe2x80x9d; Proc. Soc. Exp. Biol. Med. 131: 871-875 (1969)). This principle was adopted by Schifreen et al., xe2x80x9cA quantitative automated immunoassay for fibrinogen/fibrin degradation productsxe2x80x9d, Clin. Chem. 31: 1468-1473 (1985), with the erythrocytes being replaced by latex particles.
Other aggregation assays for determining fibrin(ogen) degradation products utilize latex particles which are coated with antibodies against fibrin(ogen) degradation products. The known antibodies were produced by immunizing with the native degradation products. Antibodies having a variety of specificities were employed.
It is a feature possessed in common by assays which use polyclonal antibodies or fibrinogen receptors that cross-reactivity reactivity with fibrinogen exists. As a result of the sample pretreatment which is necessary in the assays, the samples contain different quantities of fibrinogen and artificially produced cleavage products, so that these methods at best permit semiquantitative conclusions (Gaffney P. J. and Perry M. J., xe2x80x9cUnreliability of current serum degradation products (FDP) assaysxe2x80x9d, Thromb. Haemost. 53: 301-302 (1985); Nieuwenhuizen W., xe2x80x9cPlasma assays of fibrinogen/fibrin degradation products and their clinical relevancexe2x80x9d, in: Fibrinogen 2, Biochemistry, Physiology and Clinical Relevance. G.D.O. Lowe et al., Edt., 173-180 (1987)).
It is true that, owing to the specificity of the antibodies, assays which use monoclonal antibodies also avoid the problems of cross-reactivity with intact fibrinogen or fibrin. However, for use in agglutination assays, the detected epitope must be available to the antibodies twice on the antigen in order to form aggregates. For this reason, the abovementioned latex assays, for example, which use monoclonal antibodies against D monomer (e.g. Patent Application WO 86/01298), recognize D dimer, which only arises from fibrin following cross-linking, and not D monomer (see Gaffney P. J. et al., xe2x80x9cMonoclonal antibodies against fibrinogen, fibrin and their fragments.xe2x80x9d, Thromb. Haemost. 54: 733-734 (1985)). These tests are thus not suitable for detecting fibrin(ogen)degradationproducts.
In addition to these homogeneous tests, an ELISA has been described recently in which it is possible to differentiate between fibrinogen and fibrin cleavage products with the aid of monoclonal antibodies (Koppert P. W. et al., xe2x80x9cA monoclonal antibody-based enzyme immunoassay for fibrin degradation products in plasmaxe2x80x9d, Thromb. Haemostas. 59: 310-315 (1988)).
However, as compared with homogeneous methods, the known ELISA methods are, for fundamental reasons, more labor-intensive and more time-consuming and, as a rule, more difficult to automate.
The use of a hexapeptide from the N terminus of the xcex1 chain of fibrin, arising under the influence of thrombin, is disclosed in DE 37 01 812.
Hui K. Y. et al. (xe2x80x9cMonoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogenxe2x80x9d, Science 222: 1129-1132 (1983)) used a heptapeptide from the corresponding N terminus of the xcex2 chain. The antibodies obtained by these methods recognize only fibrin and do not recognize any fibrin(ogen) cleavage products. These assays, and also the detection of the A and B fibrinopeptides which are released during the conversion by thrombin of fibrinogen to fibrin, can be employed for diagnosing hypercoagulatory, but not hyperfibrinolytic, conditions.
Specific antibodies, which react exclusively with fragment E and do not recognize the native fibrinogen or fibrin, are necessary for detecting fibrin(ogen) cleavage products in human blood, synovial fluid or urine. Moreover, these antibodies should be easy to obtain and be usable in all known immunochemical methods, i.e. both heterogeneous and homogeneous test methods.
The present invention was therefore based on the object of making available an antigen which leads to the formation of antibodies against the cleavage products of fibrinogen and fibrin, which antibodies are easy to purify and are specific and thus render possible exact quantification of the fibrinolytic activity in biological fluids, independently of the content of fibrinogen or fibrin. Furthermore, these antibodies should also make possible the use of homogeneous immunoassay techniques in addition to heterogeneous assays.
It has been found, surprisingly, that, by immunizing animals with synthetic peptides from the C-terminal regions of fragment E, antibodies can be obtained which, while not reacting with fibrinogen or fibrin, react specifically with all 3 E fragments, and, in addition, can be used in agglutination assays.
The invention therefore relates to synthetic peptides which possess amino acid sequences which correspond at least in part to the carboxy terminal ends of the E fragment, which ends arise as a consequence of the cleavage of fibrinogen by plasmin, and are antigenic; preferably, they contain at least one of the following amino acid sequences:
a) Leu-Phe-Glu-Tyr-Gln-Lys-OH(SEQ ID NO: 1),
b) Tyr-Met-Tyr-Leu-Leu-Lys-OH(SEQ ID NO: 2),
c) Val-Lys-Glu-Leu-Ile-Lys-OH(SEQ ID NO: 3) and
d) His-Gln-Val-Glu-Asn-Lys-OH(SEQ ID NO: 4),
particularly preferably, at least one of the following amino acid sequences:
a) Asn-Lys-Leu-Lys-Asn-Ser-Leu-Phe-Glu-Tyr-Gln-Lys-OH(SEQ ID NO: 5),
b) Ser-Ser-Ser-Ser-Phe-Gln-Tyr-Met-Tyr-Leu-Leu-Lys-OH(SEQ ID NO: 6),
c) Glu-Asn-Lys-Thr-Ser-Gln-Val-Lys-Gln-Leu-Ile-Lys-OH(SEQ ID NO: 7) and
d) Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys-OH(SEQ ID NO: 8).
Very particularly preferably, at least one of the following amino acid sequences:
a) His-Gln-Ser-Ala-Cys-Lys-Asp-Ser-Asp-Trp-Pro-Phe-Cys-Ser-Asp-Glu-Asp-Trp-Asn-Tyr-Lys-Cys-Pro-Ser-Gly-Cys-Arg-Met-Lys-Gly-Leu-Ile-Asp-Glu-Thr-Asn-Arg-Ile-Asn-Lys-Leu-Lys-Asn-Ser-Leu-Phe-Glu-Tyr-Gln-Lys-OH (SEQ ID NO: 9) (peptide 1)
b) Lys-Val-Glu-Arg-Lys-Ala-Pro-Asp-Ala-Gly-Gly-Cys-Leu-His-Ala-Asp-Pro-Asp-Leu-Gly-Val-Leu-Cys-Pro-Thr-Gly-Cys-Gln-Leu-Gln-Glu-Ala-Leu-Leu-Ile-Arg-Asn-Ser-Val-Asp-Glu-Leu-Asn-Asn-Asn-Val-Glu-Ala-Val-Ser-Gln-Thr-Ser-Ser-Ser-Ser-Phe-Gln-Tyr-Met-Tyr-Leu-Leu-Lys-OH (SEQ ID NO: 10) (peptide 2)
c) Tyr-Val-Ala-Thr-Arg-Asp-Asn-Cys-Cys-Ile-Leu-Asp-Glu-Arg-Phe-Gly-Ser-Tyr-Cys-Pro-Thr-Thr-Cys-Gly-Ile-Ala-Asp-Phe-Leu-Ser-Thr-Tyr-Gln-Thr-Leu-Gln-Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys-Th -Ser-Glu-Val-Lys-Gln-Leu-Ile-Lys-OH (SEQ ID NO: 11)(peptide 3) and
d) Tyr-Val-Ala-Thr-Arg-Asp-Asn-Cys-Cys-Ile-Leu-Asp-Glu-Arg-Phe-Gly-Ser-Tyr-Cys-Pro-Thr-Thr-Cys-Gly-Ile-Ala-Asp-Phe-Leu-Ser-Thr-Tyr-Gln-Thr-Leu-Gln-Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys (SEQ ID NO: 12) (peptide 4).
Peptides are also preferred which are composed of the amino acid sequences corresponding to peptides 1-3 (SEQ ID NO: 9-11).
The peptides according to the invention may also be bound to a carrier molecule, either directly or via a spacer.
They are prepared by genetic manipulation or by chemical synthesis.
The invention additionally relates to antibodies which react immunochemically with the peptides according to the invention.
These antibodies are obtained by immunizing an animal with a peptide according to the invention.
Particularly if they are polyclonal, the antibodies according to the invention are preferably isolated and purified by being immunoabsorbed to peptides according to the invention.
The antibodies according to the invention may also preferably be monoclonal antibodies which are prepared by processes known to the person skilled in the art.
The invention also relates to diagnostic methods for the immunochemical determination of intramolecular oligomers of cleavage products of fibrinogen and fibrin, using peptides according to the invention and/or antibodies according to the invention, in particular for determining fibrin(ogen) E fragments and D dimers.
In this context, a heterogeneous immunoassay is preferred, particularly preferably an enzyme immunoassay.
A homogeneous immunoassay is also preferably used, particularly preferably a particle-boosted, nephelometric or turbidimetric test.
In the method, one part of the antibodies is advantageously bound to a solid phase with the other part carrying a detectable function, where a method is preferred which uses microtitration plates as the solid phase and the detectable funtion is a fluorogenic or luminescent dye or an enzyme.
In the homogeneous immunoassay, a particulate, water-in-soluble support is advantageously used as the solid phase and the agglutination reaction is measured nephelometrically or turbidimetrically.
In heterogeneous and homogeneous methods, only one monospecific species is advantageously used, a method also being advantageous, however, in which the capture antibody(ies) is/are an antibody(ies) according to the invention, while the detection antibody(ies) can be different therefrom.
The invention also relates to the use of peptides according to the invention for therapeutic purposes, in particular for the therapy of disturbances of the fibrinolytic system.
The invention further relates to the use of antibodies according to the invention for therapeutic purposes, in particular for the therapy of disturbances of the fibrinolytic system.
In addition to this, the invention relates to an immunochemical method for determining intramolecular oligomers, in which method only one monospecific antibody species is used.
The method may be a heterogeneous assay, preferably an enzyme immunoassay, or a homogeneous immunoassay, preferably a particle-boosted, nephelometric or turbidimetric test.
In the heterogeneous immunoassay, one part of the antibodies is advantageously bound to a solid phase with the other part carrying a detectable function.
In this context, a microtitration plate is preferably used as the solid phase and the detectable function is a fluorogenic or luminescent dye or an enzyme.
Without thereby wishing to stipulate a particular mechanism of action, the assumption appears justified that a lysine at the carboxyterminal end is of importance for the peptides according to the invention.
The peptides according to the invention may be prepared by processes which are known per se to the person skilled in the art, for example (Example 1) protected amino acid derivatives or peptide segments can, in this context, be coupled to each other in solution or on a solid phase, and peptides according to the invention obtained by eliminating the protective groups and, in the case of a solid phase, by cleavage from the carrier resin. In this context, the Fmoc group is preferably used as the temporary protective group, and t-butyl/Boc-based groups for the side groups, the Pmc or Mtr group for Arg, and the tert-butylmercapto or trityl groups for Cys, are preferably used as the permanent protective groups. The C-terminal amino acid is immobilized by way of p-alkoxy-benzyl ester groups which are bound to a polymeric support which is customarily suitable for peptide synthesis, preferably crosslinked polystyrene. The peptide synthesis is effected with the repeated elimination of Fmoc, preferably using 20% piperidine in DMF (dimethyl-formamide) (v/v), and coupling the subsequent, protected amino acid, preferably using a carbodiimide in the presence of HOBT. For this purpose, the amino acid derivative is coupled in an excess, preferably 3-fold, for 1-1.5 h in DMF. After each procedural step, Fmoc elimination or condensation step, the resin is washed 3 times on each occasion with small (15 ml/g) portions of DMF or isopropanol. The peptides according to the invention are cleaved off by acidolysis, with the side chain groups being liberated at the same time. If appropriate, sulfhydryl groups which are to be uncovered are xe2x80x9cdeprotectedxe2x80x9d with tri-n-butylphosphine in an alcohol, for example trifluoroethanol, or with DTT in water. In the case of Cys (TrT) deprotection, a separate procedural step using ethanedithiol as a scavenger is unnecessary. The peptides can be purified, for example, by ion exchange chromatography, reversed-phase chromatography and gel permeation chromatography. The correct composition of the peptides and the peptide contents are determined by amino acid analysis.
Antibodies which are directed against a peptide or polypeptide which corresponds to the region of the xcex3 chain of fibrinogen from amino acid 1 to 62 react specifically with the different forms of the E fragment (see also Example 7). Both the complete polypeptides from the said regions and component sequences of these peptides are suitable for the immunization. A particularly preferred embodiment provides for the use of octadecapeptides, for example having the sequences Val-Asp-Lys-Asp-Leu-Gln-Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys (SEQ ID NO: 13) from the C terminus of the xcex3 chain of the E fragment.
For the said cases, it is important that the carboxy terminal sequence of the molecule is exposed and leads to the immunization.
In view of the use planned for the peptides, it is sensible to introduce amino acids possessing reactive side groups into the peptides in such a way that they do not affect the structure of the hapten. For this reason, cysteine, whose free SH group is suitable for coupling via thioether to many carriers, is, where appropriate, expediently added to the N-terminal end. For example, the antigen represented by the abovementioned peptide is preferably made available in the form of the nonadecapeptide Cys-Val-Sp-Lys-Asp-Leu-Gln-Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys (SEQ ID NO: 14).
The peptides employed for the immunization can be prepared by chemical synthesis in a manner known per se to the person skilled in the art as well as by purifying a polypeptide prepared by genetic manipulation, or by purifying a peptide which was obtained biochemically from fragment E by the action of proteases and/or chemically by the action of reagents, such as, for example, cyanogen bromide, which cleave peptide chains.
Peptides which are to be employed for the immunization or are to be used as immunoadsorbents are usefully coupled to a carrier molecule. Coupling processes are known per se to the person skilled in the art and are described in the literature (Nakane, P. K. et al., xe2x80x9cPeroxidase-Labeled Antibodyxe2x80x94A New Method of Conjugationxe2x80x9d, J. Histochem. Cytochem, 22: 1084-1091 (1974), Freifelder, D. M., xe2x80x9cPhysical Biochemistry,xe2x80x9d W. H. Freeman and Co. 1976). Carrier molecules within the meaning of this invention may be: natural or synthetic macromolecules as used by the person skilled in the art for producing an immunore-active conjugate, for example albumin, ovalbumin, keyhole limpet hemocyanins or polysaccharides. In a preferred embodiment, the peptide or polypeptide is bound to keyhole limpet hemocyanin.
When using the synthetic peptides according to the invention as immunoadsorbents, it is advisable to couple them to materials which are suitable for preparing solid matrices. Carrier molecules in this sense are insoluble polymers as used by the person skilled in the art for immobilizing proteins and peptides, such as, for example, polystyrene, nylon, agarose or magnetizable particles. In this context, the solid phase can be present in any desired form, for example as small tubes, nonwoven fabric, spheres, fibers or microparticles.
A preferred embodiment provides for the coupling of peptides, for example the abovementioned nonadecapeptide, to cyanogen bromide-activated Sepharose.
The immunization of appropriate animals with carrier-bound peptides leads reproducibly to the formation of antibodies. In this context, a preferred animal species for the immunization and isolation of antibodies is the rabbit; in addition to this, mice may also be used for the immunization.
The immunoglobulin fraction which is relevant for specific tests can be enriched by customary immunoadsorptive methods from such an antiserum which has been produced in an animal using synthetic peptides in accordance with the invention. However, in this case, it is preferred likewise to use a peptide which is coupled to a carrier and which possesses the same antigenic determinant as the peptide employed for the immunization as the material for such a matrix employed for the immunoadsorption. The peptide used for the immunoadsorptive purification may also have a truncated amino acid sequence; the sole prerequisite for its use in the immunoadsorptive purification of the desired antibody is that the antigenic determinant formed by this abbreviated polypeptide is recognized and efficiently bound by the desired antibody.
The peptide used for the immunoadsorptive isolation of the antibodies may be, for example, a nonadecapeptide; the peptide Cys-Val-Asp-Lys-Asp-Leu-Gln-Ser-Leu-Glu-Asp-Ile-Leu-His-Gln-Val-Glu-Asn-Lys (SEQ ID NO: 14) is preferred. In accordance with the invention, antibodies are induced in an animal system by immunizing with synthetic peptides and then purified, where appropriate, by immunoadsorption. These antibodies react specifically with the peptides used for the immunization and purification.
By choosing appropriate peptides as immunoadsorbents, antibodies can be selected which preferably react specifically with the antigenic determinant of fragment E which corresponds to the plasmin cleavage site of this molecule. In the preferred case, where peptides possessing sequences from the C-terminal region of the plasmin recognition sequence are used both for the immunization and for the immunoadsorptive purification, antibodies against these sequences are enriched.
Monoclonal antibodies having the properties according to the invention may also advantageously be prepared by processes which are known per se to the person skilled in the art.
The antibodies isolated according to the invention can be employed for homogeneous and heterogenous immunoassays, such as, for example, enzyme immunoassays or free or particle-boosted agglutination reactions, which are known per se to the person skilled in the art. Preferably, they are coupled for this purpose to a solid support. Natural and synthetic, organic and inorganic, polymers, which are known per se to the person skilled in the art, are suitable solid, water-insoluble supports; examples are: polystyrene, polydextrans, polypropylene, polyvinyl chloride, polyvinylidene fluoride, polyacrylamide, agarose, latex, magnetite, porous glass powder, erythrocytes, leucocytes, blood platelets or copolymers consisting of styrene-butadiene, styrene-methacrylic acid or methacrylate-methacrylic acid. Tubes, spheres or microtitration plates are suitable geometric embodiments.
In a preferred manner, the content of fragment E is determined in accordance with the invention by incubating the sample with antibodies which are immobilized on particulate supports, the concentration of fragment E bound by the immobilized antibodies being detected turbidimetrically or nephelometrically by way of the turbidity arising under these circumstances.
In accordance with the invention, the concentration of the D dimer/E complex can also be determined using an antibody immobilized in this way. The prerequisite is the use of a specific antibody against D dimer as a second antibody which is immobilized either on the same or on different particles. One of the two antibodies may also be present free in solution such that immunocomplexes of the configuration: particle-antibody 1/antigen/free antibody 2/antigen/particle-antibody 1 are formed and are quantified nephelometrically or turbidimetrically.
In addition, heterogeneous detection methods are preferred in which antibodies according to the invention are immobilized on the solid phase in the ELISA technique. Fragment E or the D dimer/E complex is bound to the immobilized antibody in a first incubation step. The bound antigen is detected in a second incubation step using the same or a different antibody. This second antibody must possess a property which is measurable, for example the ability to convert or bind a chromogenic substrate.
The second antibody can be provided, for example, with an enzyme, a fluorescent molecule, such as, for example, fluoroscein isothiocyanate, a radioactive label, or a molecule which is capable of chemiluminescence. Preferably, this second antibody is coupled to a marker enzyme; peroxidase is particularly preferred.
Fragment E or D dimer/E complex can also be determined by simultaneously incubating the sample, preferably of plasma, and labeled antibody together with the immobilized antibodies. In addition to this, a competitive determination method is possible in which labeled and unlabeled fragment E or D dimer/E complex compete for the binding site of the immobilized antibodies. The content of fragment E or D dimer/E complex determined in this way permits conclusions to be drawn with regard to the degree of activation of the fibrinolytic system.
The embodiments specified in the examples are particularly preferred. The examples illustrate the invention without, however, limiting it in any way.
The following abbreviations are used:
ELISA enzyme immunoassay (enzyme linked immunosorbent assay)
KLH keyhole limpet hemocyanin
PBS phosphate-buffered sodium chloride solution (phosphate buffered saline)
Tris tris(hydroxymethyl)aminomethane
OD extinction (optical density)
Cys cysteine
Amino acids can be present in the D or L configuration; unless otherwise indicated they are present in the L form.
Val valine
Asp aspartic acid
Lys lysine
Cys cysteine
Leu leucine
Gln glutamine
Ser serine
Glu glutamic acid
Ile isoleucine
His histidine
Asn asparagine
Boc t-butoxycarbonyl
Fmoc 9-fluorenylmethoxycarbonyl
Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl
DMF dimethylformamide
HOBt hydroxybenzotriazole
DTT dithiothreitol
Trt trityl